We report on the results of the multicenter evaluation of the CEDIA Theophylline assay on Boehringer Mannheim/Hitachi analyzers in 15 clinical laboratories in Europe and U.S.A. Main items of investigation were imprecision, recovery of control sera, interlaboratory survey and method comparisons using patient samples. Imprecision was found to be comparable to other routine methods. An advantage of the CEDIA assay can be seen in the good interlaboratory transferability of results. The new test has been shown to measure very accurately particularly by comparison with HPLC procedures revealing highly correspondent results. The reagent can be used up to one month using multiple recalibration. Due to its high practicability and reliability the CEDIA Theophylline assay can be recommended as a very suitable routine method for therapeutic drug monitoring on random access analyzers like Boehringer Mannheim/Hitachi analysis systems.
The sensitivity of a new homogeneous enzyme immunoassay for the determination of digoxin (CEDIA Digoxin assay) and a fluorescence polarization immunoassay (FPIA) to interference by digoxin-like immunoreactive factors (DLIF) was studied in sera from pregnant women, newborns, patients undergoing hemodialysis and patients with renal insufficiency, but without hemodialysis. None of the patients had been treated with digoxin or digitoxin. Cross-reactivity of DLIF in the CEDIA assay was generally lower than in the FPIA. Data on the distribution DLIF of values and method comparisons showed that sera of the four patient groups reacted in a completely different way in both assays, suggesting that the nature of DLIF in the four groups is not identical. Addition of digoxin to sera of patients not treated with this drug resulted in a reduction of the apparent DLIF concentration in the CEDIA assay and the FPIA. This shows that DLIF interference may be less pronounced in sera of patients undergoing digoxin therapy compared to untreated persons. Although the CEDIA assay is less sensitive to DLIF interference than the FPIA, further efforts are needed to reduce the extent of this interference.
Serum cortisol is one of the more frequently requested steroid hormone assays. Its use is important in evaluating diseases of the adrenal cortex and pituitary. We briefly review the biochemistry of cortisol synthesis, the pathophysiology resulting from adrenal and pituitary abnormalities and the more specific immunochemical procedures which have replaced colorimetric chemical assays for cortisol. We also report our results on the evaluation of the analytical performance of the non-isotopic homogeneous CEDIA Cortisol assay and compare the advantages of this assay to state-of-the-art immunoassays.
The overall reliability of measuring digoxin in serum improved significantly with the discovery and application of immunoassays. However, because of the low concentration of digoxin being measured, its narrow therapeutic range in serum, and the presence of endogenous digoxin-like immunoreactive factors (DLIF), developing assays for measuring digoxin still pose formidable challenges. In this presentation, recent developments in the characterization of DLIF from bovine adrenal cortex and human serum are described. Data accumulated to date suggest there is one principal endogenous molecular factor (DLIF) in humans that cross-reacts with anti-digoxin antibodies. This factor exists at sufficiently high concentrations in some patients to interfere with measurements of digoxin by most digoxin immunoassays. All digoxin immunoassays should be tested to interference from this endogenous factor. Various techniques for reducing DLIF cross-reactivity are reviewed. The isolation and purification of DLIF now provides new approaches for selecting specific anti-digoxin antibodies used in developing more accurate digoxin immunoassays.
In the last two decades two major trends heavily influenced the situation of the clinical laboratories. Cost saving issues have become more and more significant and an exploding number of tests has to be performed by a limited number of technicians. The latter task is facilitated by the use of automated analyzers. The CEDIA assays for therapeutic drug monitoring (TDM) can be performed on Boehringer Mannheim/Hitachi systems used for routine clinical chemistry. This means that now it is possible to determine all parameters of clinical chemistry, proteins, and TDM without sample splitting on one analyzer, resulting in saving of manual workload and costs.
The CEDIA Phenobarbital assay has been evaluated in twelve clinical laboratories in Europe and U.S.A. on Boehringer Mannheim/Hitachi analysis systems. The evaluation focused on the analysis of imprecision and accuracy. Within-run and between-day coefficients of variations of the new assay were comparable to those of established routine methods. As demonstrated in an interlaboratory survey study with controls and human sera, results obtained in different laboratories showed a good agreement. The CEDIA Phenobarbital assay measured very accurately, as particularly confirmed by comparison with HPLC. It can be recommended as a reliable and practicable test for monitoring of phenobarbital on Boehringer Mannheim/Hitachi analyzers used in routine clinical chemistry.
We evaluated a CEDIA(R) assay for the determination of digitoxin in serum on random access analyzers. The multicenter evaluation included studies on the analytical range, calibration stability and reproducibility of the new assay. Moreover, recovery in controls, transferability of results obtained in different laboratories, comparability with routine methods, and the effect of various interfering factors have been analyzed. Summarized the analytical performance was comparable to that of routine methods. The CEDIA(R) Digitoxin assay represents an attractive alternative to established digitoxin immunoassays because it can be performed on random access analyzers, thus permitting the simultaneous determination of digitoxin and other serum analytes without sample splitting.
Sera from the routine of therapeutic drug monitoring were assayed for phenobarbital, phenytoin, and theophylline with three different methods: fluorescence polarization immunoassay as the standard procedure, the new CEDIA assays within a multicenter evaluation and HPLC which is known to yield results with a high specificity. CVs for between-day imprecision ranged from 2.6-8.6%, depending on the concentration of the drugs. There was a tendency to lower CVs for the HPLC procedure. Accuracy was verified with commercial control materials and spiked sera and proved to be satisfactory for all three methods and parameters. The linear range was approx. twice as wide for the HPLC compared with the other methods. The method comparisons were quite favorable. Deviations occurred mainly in the subtherapeutic concentration range.
A new CEDIA assay for the measurement of digoxin in serum on random access analyzers was evaluated by twelve laboratories in Europe and the United States. Studies on the analytical range, reproducibility, calibration stability, recovery in controls, interlaboratory comparability, comparability with routine methods, and the effect of various interfering factors have been performed and the results are presented in this paper. The analytical performance was comparable to that of routine methods provided the manual pipetting step for pre-incubation was performed with accurate pipettes. A major advantage of the CEDIA Digoxin assay in terms of convenience is the simple two-point calibration procedure. Moreover, digoxin can be determined within 15 minutes after receiving the samples on random access analyzers like Boehringer Mannheim/Hitachi analysis systems. Thus, the CEDIA Digoxin assay represents an attractive alternative to the measurement of digoxin on dedicated immunochemical assay systems.
New homogeneous enzyme immunoassays have been developed for cortisol, digoxin, digitoxin, theophylline, phenytoin, and phenobarbital using the cloned enzyme donor immunoassay technology. As applied to Boehringer Mannheim/Hitachi analysis systems these methods provide rapid, accurate and precise quantification of analytes, with minimal interferences from endogenous serum constituents and low cross-reactivities to structurally-related hormonal precursors, drug metabolites and natural compounds. Additional significant features of the new assays are linear standard curves and two-point calibration. The six CEDIA assays join the two currently available CEDIA assays for determination of the thyroid parameters T4 and T Uptake. Additional new therapeutic drug and anemia monitoring assays are under development, demonstrating the versatility of the cloned enzyme donor immunoassay technology. These tests, in concert with Boehringer Mannheim/Hitachi analyzers, provide a high throughput, random access immunoassay system. The menu of available assays should continue to increase during the 1990s, providing efficient automation while allowing consolidation of testing on a limited number of instrument systems.
Various factors known to affect digoxin blood values are discussed in the present review. A reduction as well as an increase in digoxin levels may be due to the method employed, to errors in the preanalytical phase, to peculiarities of the patient, to diseases or to the administration of drugs. Increased digoxin concentrations above the therapeutic range are much more common than manifestations of digoxin intoxication. However, geriatric patients are specially sensitive to intoxication. Attention has to be paid to digoxin-like immunoreactive factors (DLIF) being synthesized under certain physiologic and pathophysiologic conditions which cause falsely elevated values in digoxin-immunoassays. The determination of digoxin levels is indicated in suspected intoxication or hypersensitivity to digoxin, in patients with pacemakers or those receiving certain drugs, in old people and in patients suffering from any concomitant disease. The time of blood collection has to be carefully controlled in order to determine digoxin concentrations of diagnostic value, the physical activity of the patient being a main influencing factor.
Some aspects of a new homogeneous enzyme immunoassay for the determination of digoxin have been evaluated, as part of a multicenter project. The CEDIA Digoxin assay is based on the use of two beta-galactosidase fragments (EC 3.2.1.23) produced by recombinant DNA techniques, one of them linked to digoxin. These two fragments couple to form the complete active enzyme, if not hindered by anti-digoxin antibodies. Digoxin in serum competes for antibody binding. The procedure can easily be automated and does not require special equipment. The imprecision of the method was studied at three different concentration levels (0.56, 1.29 and 2.77 ng/mL of digoxin). Within-run coefficients of variation were 8.01%, 5.57% and 3.30%, respectively, the corresponding between-day CVs being 17.4%, 8.41% and 4.89%. The procedure was found to be linear up to 4.4 ng/mL. Reagents were stable for at least four weeks. Results obtained by the CEDIA Digoxin assay compared well with those obtained by fluorescence polarization immunoassay.
Opinions on the clinical relevance of the determination of antiepileptic drugs in serum are very divergent. Specially the value of routine determinations has not been clarified up to now. At present the determination of antiepileptic drugs is indicated in the following situations: resistance to therapy including suspected irregular intake, adjustment of the serum level into the therapeutic range to prevent under- or overdosage, suspected intoxication (particularly during combined therapy), dosage monitoring when changes in the dosage-serum level relationship might occur, combined therapy, dosage reduction in the absence of seizures, determination of a baseline level in patients with good anticonvulsant control, legal indications, therapy research.
The need for monitoring serum and plasma concentrations for therapeutic drugs given to hospitalized and outpatients will continue to growth during the next few years, both in terms of the number of tests ordered each year and the number of different drugs that are monitored. The clinical laboratory will play an important role in implementing and maintaining accurate, cost-effective drug monitoring and treatment programs. To meet changing needs, further development and evaluation of automated analytical assays will be necessary.
Therapeutic drug monitoring is recommended in suspected drug overdosage, in cases of therapeutic failure, in the establishment of regimens in patients with altered pharmacokinetics and in patients with unknown previous medications. Pharmacokinetic dose prediction methods have been developed allowing individual dosage adaptation. As examples, the three-point method of Sawchuk and the Bayesian technique are described. Drug concentrations could be predicted with sufficient accuracy by use of the three-point method in critically ill patients treated with aminoglycosides or flucytosine. Bayesian forecasting proved to be useful in predicting the nocturnal concentration curves in patients with asthma treated with a sustained-release preparation of theophylline for once-daily dosage. The efficiency of drug treatment can be considerably improved by monitoring serum drug concentrations and individualizing dosage using pharmacokinetic methods.
New homogeneous enzyme immunoassays (CEDIA assays) for therapeutic drug monitoring were evaluated on Boehringer Mannheim/Hitachi 704. A fluorescence polarization immunoassay and HPLC were chosen as comparison methods. A good correlation of patient data was observed for both methods (slopes 1.033-1.167). Imprecision within-run and between-day as well as the recovery in control samples of an external quality control survey were excellent and comparable to the routine method. In order to optimize the use of the CEDIA assays also for parameters which are less frequently requested, a parameter-setting for the batch-analyzer COBAS Fara was worked out. Performance of the CEDIA assays on random access analyzers is discussed with regard to the background of a laboratory for clinical chemistry in a University Hospital.
In clinical chemistry two different quantities are determined for electrolytes: 1) Electrolyte concentration (total) in serum (S) e.g. S-sodium (mmol/l), S-calcium (mmol/l). 2) Electrolyte concentration (ionized) in serum water [S(W)] e.g. S(W)-sodium, ionized (mmol/kg), S(W)-calcium, ionized (mmol/kg) ad 1) For the determination of the electrolyte concentration in serum, various methods are used: Sodium, potassium: Flame atomic emission spectrometry, ion-selective electrodes after dilution of the sample, enzymatic methods; Chloride: Coulometry, absorption spectrometry after chemical reaction, enzymatic method; Calcium, magnesium: Flame atomic absorption spectrometry, flame atomic emission spectrometry (calcium), absorption spectrometry after chemical reaction, enzymatic method (magnesium). A safe and unambiguous medical interpretation of sodium and chloride ion concentration in serum is not possible without knowledge of the water concentration or of the lipid and protein concentration of the individual sample. The same holds true--even though for some other reasons--for calcium concentration in whole serum. The reference intervals of the pertinent ions are valid only for samples, which are "normal" with respect to the size of the electrolyte-free compartment and--depending on the method--the amount of complex-binding ions. ad 2) For the determination of the concentration of the "ionized" or "free" fraction of sodium, potassium, calcium, and magnesium in serum water (or the extracellular water phase of whole blood) the following method is only applicable: Ion selective electrode without dilution of the sample. A save medical interpretation of the ionized electrolyte concentration in serum water is possible without knowledge of the water concentration of the individual sample, because these quantities are independent from the size of the electrolyte-free compartment.(ABSTRACT TRUNCATED AT 250 WORDS)
The principles and main features of enzymatic methods for the measurement of sodium, potassium, and chloride are reviewed and their performance compared with current procedures. Each method makes use of a relatively specific enzyme, catalysing a reaction whose rate is sensitive to the ion to be determined. Where the (S)0.5 of the enzyme for the ion is much lower than the assay concentration, the ion concentration may be reduced by a binding agent. Alternatively, a competitive inhibitor may be used to raise the (S)0.5 of the enzyme. In the case of chloride determination with amylase the (S)0.5 of the enzyme is raised by limiting the concentration of free calcium. In the measurement of potassium, interfering ions such as sodium are removed by binding with Kryptofix 221 and improvement in performance is also achieved by use of a bacterial pyruvate kinase less sensitive to sodium. The enzymatic methods are applicable to measurement of sodium, potassium and chloride in blood or urine with good precision, accuracy, and specificity. They can be used on mechanized or manual instruments. There appears to be minimal interferences from compounds found in normal or pathological serum or urine.
